The goals of this proposal are twofold: the creation of a versatile genetic system for manipulating the fate of mouse embryonic stem cells and the use of this system to begin to investigate the signaling pathways that regulate early definitive endoderm development. Stem cells have received much attention as potential sources for novel therapies. By virtue of their ability to differentiate into a wide variety of different cell types, embryonic stem (ES) cells may also represent an opportunity to learn about the genetic regulation of normal embryo development. To investigate the genetic control of ES cell differentiation, tools permitting regulated gene expression in ES cells are required. To this end, we are developing a novel gene expression system called TetTarget for use in mouse ES cells. TetTarget builds upon the well-described Tet regulatory system to confer robust gene expression and, unlike expression systems currently employed in ES cells, provides highly specific regulation of the level of induction, the timing of induction and the cell types in which gene induction occurs. Additionally, we make use of recombination mediated cassette exchange technology to allow new genes of interest to be efficiently introduced into the TetTarget expression cassette. Thus, TetTarget is a versatile genetic system that we anticipate will be helpful to members of the developmental biology community seeking to study varied aspects of stem cell biology. We propose to use the TetTarget system to study the development of definitive endoderm. We have chosen to concentrate on endoderm development for two reasons: less is known about endoderm development than mesoderm or ectoderm development, and a better understanding of endoderm development will help to establish approaches to producing stem cell-derived hepatic and pancreatic cells. Our initial efforts will focus on evaluating the function of genes previously implicated in early endoderm development. Specifically, we will use TetTarget to express candidate endodermal regulators such as activators of the Nodal signaling pathway and members of the Sox, Gata, Forkhead and Mix transcription factor families in embryonic stem cells. We will assess whether these regulators promote definitive endoderm formation and how changing the level and timing of expression affects endoderm production. Ultimately, this approach seeks to provide novel insights how ES cell differentiation into definitive endoderm is regulated, and by extension, how the definitive endoderm arises and develops in vivo. [unreadable] [unreadable]